title: Investigating citrullinated proteins in tumour cell lines.
abstract: BACKGROUND
  The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB).
  
  
  METHODS
  Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines.
  
  
  RESULTS
  2-D WB and mass spectrometry identified citrullinated α-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines.
  
  
  CONCLUSIONS
  The citrullination of these proteins suggests a new mechanism in the tumorigenic process.
authors.affiliation: Medical Research Center of Qianfoshan Hospital, Shandong University, Jingshi Road 16766, Jinan, Shandong 250014, P,R, China. changxt@126.com.
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Jiang
authors.forename: Yazhou
authors.initials: Y
authors.lastname: Cui
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Suhua
authors.initials: S
authors.lastname: Yan
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Chang
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D002956:Citrulline / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D009369:Neoplasms / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D057825:Two-Dimensional Difference Gel Electrophoresis
chemical_list: D014408:Biomarkers, Tumor; D002956:Citrulline